Plant materials and experimental design
The two-year-old seedlings were from Yangkou Forest Farm, Fujian Province. The normal cultivar were the first generation seedlings, and the super cultivar were the third generation varieties. The seedlings with relatively consistent height and diameter were planted in 30-L large plastic pots. Seedlings were grown in a controlled environment room at Zhejiang A & F University (N30°23′, E119°72′), China. Before the trial started, to keep the seedlings growing well, we irrigated the pots once every three days or when there was a need.
The experiment employed a completely randomized design with 12 factorial combinations of two Chinese fir cultivars (NC and SC), two types of soil (NP soil and CP soil), and three levels of water stress. This trial had three replications per treatment and five plants per replication (plastic pot). The Chinese fir cultivars were collected from Shaxian, Fujian province. SC and NC were 2.5 and 1.5 strain cultivar of C. lanceolata, respectively. SC has stronger ability of height growth and diameter growth than NC . The sapling stage before available for timber production is shorter than that of NC. The performance of SC in clear-cutting forestland of Chinese fir plantation was significantly better than that of sprouting trees . However, the ability to adapt to drought stress, and their performance in logging area were less studied. The NP soil was the soil from forest lands without planted Chinese fir, and most of the trees in this forest land were subtropical species of evergreen broad-leaved trees. The CP soil was the soil from a Chinese fir forest that had been replanted more than 20 years after the first fir harvest. The properties of the non-continuous planting soil (NP soil) used in this study were as follows (based on kg-1 dry soil): pH 5.47, total N 1.21 g, hydrolysable N 162.07 mg, total phosphorus 0.61 g, available phosphorus 2.1 mg, total potassium 13.69 g, organic matter 36.36 g. The properties of continuous planting soil from the Chinese fir forest land (CP soil) were as follows (based on kg-1 dry soil): pH 4.63, total N 0.92 g, hydrolysable N 127.08 mg, total phosphorus 0.43 g, available phosphorus 1.53 mg, total potassium 10.41 g, organic matter 28.22 g.
There were three drought treatments: one was under normal water control (watered and maintained at 75%−80% field water capacity, normal water control, CK); a second treatment was under medium drought stress (watered and maintained at 45%−50% field water capacity, medium water content, MWC), and the third was under heavy draught stress (watered and maintained at 20%−25% field water capacity, low water content, LWC). Water was added when the percentage was outside the specified level at 16:00−18:00 per 3 d by the weight method.
During the executing process, the potted seedlings of each test treatment were periodically rotated to minimize the influence of the growth factors of each treatment. During the last 10 d of this trial, protecting enzyme activity, lipid periodization materials, relative electrolyte conductivity, gas exchange, chlorophyll fluorescence and chlorophyll pigment content were all evaluated. After harvesting, samples were separated into leaves, stems and roots. The samples were used for the measurements of water content, biomass, total carbon (C), total nitrogen (N) and total phosphorus (P) in different organs.
Assays of height growth, biomass and water content
At the beginning, we selected 30 plants per cultivar to measure their height and biomass. The samples were separated into different organs for biomass assays. This was done again at the end of the trial. The increases in biomass, height growth and water content were calculated by subtracting the final data from the mean initial data. Water content was calculated by fresh weight minus the dry weight. The root-to-shoot ratio was calculated by root biomass and shoot biomass. Biomass was obtained by oven drying at 60°C until a constant weight was reached .
Assays of nutrient biological stoichiometry, nutrient use efficiency
The contents of C, N and P in roots and leaves were analyzed and determined. The C content was measured by an elemental analyzer (Vario Max cube CNS, Elementar, Germany). The N and P content in different organs were measured by the micro Kjeldhal and molybdenum antimony colorimetric methods, respectively [36, 37]. Nutrient biological stoichiometry in leaf, stem and root were calculated by N content/ P content. C use efficiency (CUE), N use efficiency (NUE) and P use efficiency (PUE) were calculated by the ratios of nutrient content in organs above the soil and in organs below the soil . These were to evaluate the allocation of nutrient in plant.
Determination of enzymes activity and lipid periodization
Fresh leaf samples from the third or fourth expanded leaves were collected for enzymes extraction. The samples were collected and put into boxes with ice bags. Enzymes were extracted at 4°C from approximately 0.2 g leaf samples with 100 mM phosphate buffer (pH 7.8). This buffer contained 0.1 mM MEDTA, 1% (v/v) polyvinyl pyrrolidone (PVP), 0.1 mM phenyl methyl sulphonyl fluoride (PMSF) and 0.2% (v/v) Triton X-100. Extracting solutions were centrifuged at 6,000 r/min for about 30 min. The supernatants were used for the measurements of superoxide dismutase (SOD; EC 188.8.131.52), peroxidase (POD; EC 184.108.40.206) and soluble protein.
SOD activity was assayed by the inhibition of the photochemical reduction of β-nitro blue tretrazolium chloride (NBT) [37, 39], and SOD was measured at 560 nm with a spectrophotometer (Shimadzu UV-2550, Kyoto, Japan). POD activity was measured with guaiacol as the substrate at 470 nm . Malondialdehyde (MDA) content is an essential parameter to illustrate the lipid peroxidation level, and this was determined via the thiobarbituric acid method (TBA) . Total soluble protein content was determined by the Coomassie brilliant blue method . The APX enzyme activity was determined by the ascorbic acid method.
Proline content was determined by the acid ninhydrin occlusion method . To a 0.20 g sample of leaves, 5 ml 3% sulfosalicylic acid solution was added with covering and extracted in a boiling water bath for 15 min (shaking frequently during the extraction), and then filtered in a clean test tube after cooling. The filtrate is the extract of proline. After obtaining the extract, the measurement was carried out with reference to the literature.
We selected five samples from the third or fourth fully expanded and exposed young leaves for gas exchange and chlorophyll fluorescence measurements.
Gas exchange was measured with the Li-cor 6400XT portable photosynthesis system (Li-cor Biosciences, Inc., Lincoln, USA) on sunny days during 8:30 to 11:00. In this system, the ambient CO2, light intensity, relative humidity and temperature were controlled at 400 µmol·mol-1, 1200 µmol·m-2·s-1, 60% and 28°C, respectively. The CO2 was provided by a dedicated CO2 steel cylinder for Li-cor 6400. The light was provided by an LED red-blue light chamber. The leaf in the chamber were collected and the area were measured with leaf area analysis system (CI202, CID, USA).
The photosynthetic N use efficiency (PNUE) was calculated as the ratio between the photosynthetic rate and leaf N concentration per area .
Water use efficiency
The instantaneous water use efficiency (WUEi) was determined by the ratio of maximum carbon assimilation at saturating light (Amax) to leaf transpiration rate (E). The five leaf samples per replication were selected for the measurement of water potential (Wp) with pressure tank (3005, Soilmositure Equipment Corp, USA). These were done during 2:00–4:30 before sunrise.
Five leaf samples per treatment were selected for the measurement of δ13C using an isotope ratio mass spectrometer (Delta V Advantage, Thermo Fisher Scientific, Inc., USA), combined with an elemental analyzer (Flash EA1112 HT, Thermo Fisher Scientific, Inc.). The samples were burned at high temperature to generate CO2 in the elemental analyzer. We measured the ratio of 13C content to 12C content using the mass spectrometer and calculated the ratio of δ13C after comparing with the international standard substance (Pee Dee Belnite, PDB).
Chlorophyll content and ultrastructure of chloroplasts
The samples were collected for each treatment for chlorophyll content determination. The harvested fir leaves were quickly washed and dried, and the midrib was cut. The sample was then cut into a 0.2 cm filament or a small piece and evenly mixed. The samples were weighed to the nearest 0.1 g, placed in a test tube, and 8 ml of 95% ethanol was added. The leaves were soaked in the dark for 24 hours, and shaken for three to four times in the middle of the period until they turned white. Each treatment was repeated three times .
Five samples per treatment were selected for examining the ultrastructure of chloroplasts using a transmission electron microscope (Hitachi H-7650, Hitachi, Ibaraki, Japan). The samples were processed as follows: (1) Double fixation: The specimen was first fixed with 2.5% glutaraldehyde in phosphate buffer (0.1M, pH7.0) for more than 4 h, washed three times in the phosphate buffer (0.1M, pH7.0) for 15 min at each step, then post fixed with 1% OsO4 in phosphate buffer (0.1M, pH7.0) for 1–2 h and washed three times in the phosphate buffer (0.1M, pH7.0) for 15 min at each step. (2) Dehydration: The specimen was first dehydrated by a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%) for about 15−20 min at each step, then transferred to absolute acetone for 20 min. (3) Infiltration: The specimen was placed in a 1:1 mixture of absolute acetone, and the final Spurr resin mixture for 1h at room temperature, and then transferred to a 1:3 mixture of absolute acetone and the final resin mixture for 3h and to the final Spurr resin mixture overnight. (4) Embedding and ultrathin sectioning: specimens were placed in Eppendorf tubes containing Spurr resin and heated at 70°C for more than 9h. The specimens were sectioned with a LEICA EM UC7 ultratome, and sections were stained by uranyl acetate and alkaline lead citrate for 5−10 min, respectively, and observed using a Hitachi Model H-7650 TEM.
Data were analyzed by the MANOVA procedure of the Statistic Analysis System (SAS). Fertilizer treatments and water availability were analyzed as main factors. Multiple comparisons were made using Duncan’s test at a significance level of α = 0.05.